Design, validation and performance of aspartate aminotransferase- and lactate dehydrogenase-reporting algorithms for haemolysed specimens including correction within quality specifications.

BACKGROUND: In vitro haemolysis is a major operational challenge for medical laboratories. A new experimental design was used to investigate under what conditions algorithms could be designed to report either quantitative or qualitative aspartate aminotransferase and lactate dehydrogenase results outside the manufacturer's haemolysis specifications. Quantitative corrections were required to meet prespecified quality specifications. METHODS: Twenty-five patient samples were used to design reporting algorithms and another 41 patient samples were used to validate the algorithms. Aspartate aminotransferase, lactate dehydrogenase and haemolysis index were determined using a Cobas 6000 analyser (Roche diagnostics, Mannheim, Germany). Correction factors were determined, and the accuracy of the correction was investigated. Reporting algorithms were designed based on (i) the manufacturer's cut-off for the haemolysis index, (ii) corrections within the total allowable error specification and (iii) qualitative reporting based on obtained results. The impact of the reporting algorithms was retrospectively determined by recalculating six months of aspartate aminotransferase and lactate dehydrogenase results. RESULTS: No correction for aspartate aminotransferase/lactate dehydrogenase was possible for results below the upper reference interval limit, while results equal to or greater than the upper reference interval limit could, up to mild haemolysis, be corrected within the total error criterion. All samples generated from the validated patient cohort fulfilled the set criteria. The algorithms allowed reporting 88.5% and 85.9% of otherwise unreported aspartate aminotransferase and lactate dehydrogenase results, respectively. CONCLUSIONS: An approach is presented that allows to generate and validate reporting algorithms for aspartate aminotransferase and lactate dehydrogenase compatible with prespecified quality specifications. The designed algorithms resulted in a significant reduction of otherwise unreported aspartate aminotransferase and lactate dehydrogenase results.

Targeting TGF-ß Signaling in Cancer.

The transforming growth factor (TGF)-ß signaling pathway is deregulated in many diseases, including cancer. In healthy cells and early-stage cancer cells, this pathway has tumor-suppressor functions, including cell-cycle arrest and apoptosis. However, its activation in late-stage cancer can promote tumorigenesis, including metastasis and chemoresistance. The dual function and pleiotropic nature of TGF-ß signaling make it a challenging target and imply the need for careful therapeutic dosing of TGF-ß drugs and patient selection. We review here the rationale for targeting TGF-ß signaling in cancer and summarize the clinical status of pharmacological inhibitors. We discuss the direct effects of TGF-ß signaling blockade on tumor and stromal cells, as well as biomarkers that can predict the efficacy of TGF-ß inhibitors in cancer patients.

Bcl-2 is a critical mediator of intestinal transformation.

Intestinal tumour formation is generally thought to occur following mutational events in the stem cell pool. However, active NF-κB signalling additionally facilitates malignant transformation of differentiated cells. We hypothesized that genes shared between NF-κB and intestinal stem cell (ISCs) signatures might identify common pathways that are required for malignant growth. Here, we find that the NF-κB target Bcl-2, an anti-apoptotic gene, is specifically expressed in ISCs in both mice and humans. Bcl-2 is dispensable in homeostasis and, although involved in protecting ISCs from radiation-induced damage, it is non-essential in tissue regeneration. Bcl-2 is upregulated in adenomas, and its loss or inhibition impairs outgrowth of oncogenic clones, because Bcl-2 alleviates apoptotic priming in epithelial cells following Apc loss. Furthermore, Bcl-2 expression in differentiated epithelial cells renders these cells amenable to clonogenic outgrowth. Collectively, our results indicate that Bcl-2 is required for efficient intestinal transformation following Apc-loss and constitutes a potential chemoprevention target.

ER-Stress-Induced Differentiation Sensitizes Colon Cancer Stem Cells to Chemotherapy.

Colon cancer stem cells (colon-CSCs) are more resistant to conventional chemotherapy than differentiated cancer cells. This subset of therapy refractory cells is therefore believed to play an important role in post-therapeutic tumor relapse. In order to improve the rate of sustained response to conventional chemotherapy, development of approaches is warranted that specifically sensitize colon-CSCs to treatment. Here, we report that ER-stress-induced activation of the unfolded protein response (UPR) forces colon-CSCs to differentiate, resulting in their enhanced sensitivity to chemotherapy in vitro and in vivo. Our data suggest that agents that induce activation of the UPR may be used to specifically increase sensitivity of colon-CSCs to the effects of conventional chemotherapy.

Cancer stem cells--important players in tumor therapy resistance.

Resistance to tumor therapy is an unsolved problem in cancer treatment. A plethora of studies have attempted to explain this phenomenon and many mechanisms of resistance have been suggested over recent decades. The concept of cancer stem cells (CSCs), which describes tumors as hierarchically organized, has added a new level of complexity to therapy failure. CSCs are the root of cancers and resist chemo- and radiotherapy, explaining cancer recurrence even many years after therapy is ended. This review discusses briefly CSCs in cancers, gives an overview of the role of CSCs in therapy resistance, and discusses the potential means of targeting these therapy-resistant tumor cells.

Mutations in the Ras-Raf Axis underlie the prognostic value of CD133 in colorectal cancer.

PURPOSE: High expression of cancer stem cell (CSC) marker CD133 has been used as a predictor for prognosis in colorectal cancer (CRC), suggesting that enumeration of CSCs, using CD133, is predictive for disease progression. However, we showed recently that both CD133 mRNA and protein are not downregulated during differentiation of colon CSCs, pointing to an alternative reason for the prognostic value of CD133. We therefore set out to delineate the relation between CD133 expression and prognosis. EXPERIMENTAL DESIGN: A CRC patient series was studied for expression of CD133 and other CSC markers by microarray and quantitative PCR analysis. In addition, several common mutations were analyzed to determine the relation with CD133 expression. RESULTS: CD133 mRNA expression predicted relapse-free survival in our patient series, whereas several other CSC markers could not. Moreover, no correlation was found between expression of other CSC markers and CD133. Interestingly, high CD133 expression was related to mutations in K-Ras and B-Raf, and inhibition of mutant K-Ras or downstream mitogen-activated protein kinase kinase (MEK) signaling decreases CD133 expression. In addition, an activated K-Ras gene expression signature could predict CD133 expression in our patient set as well as data sets of other tumor types. CONCLUSION: CD133 expression is upregulated in CRC tumors that have a hyperactivated Ras-Raf-MEK-ERK pathway and is therefore related to mutations in K-Ras or B-Raf. As mutations in either gene have been related to poor prognosis, we conclude that CD133 expression is not indicative for CSC numbers but rather related to the mutation or activity status of the Ras-Raf pathway.

Targeting colorectal cancer stem cells with inducible caspase-9.

Colorectal cancer stem cells (CSCs) drive tumor growth and are suggested to initiate distant metastases. Moreover, colon CSCs are reportedly more resistant to conventional chemotherapy, which is in part due to upregulation of anti-apoptotic Bcl-2 family members. To determine whether we could circumvent this apoptotic blockade, we made use of an inducible active caspase-9 (iCasp9) construct to target CSCs. Dimerization of iCasp9 with AP20187 in HCT116 colorectal cancer cells resulted in massive and rapid induction of apoptosis. In contrast to fluorouracil (5-FU)-induced apoptosis, iCasp9-induced apoptosis was independent of the mitochondrial pathway as evidenced by Bax/Bak double deficient HCT116 cells. Dimerizer treatment of colon CSCs transduced with iCasp9 (CSC-iCasp9) also rapidly induced high levels of apoptosis, while these cells were unresponsive to 5-FU in vitro. More importantly, injection of the dimerizer into mice that developed a colon CSC-iCasp9-induced tumor resulted in a strong decrease in tumor size, an increase in tumor cell apoptosis and a clear loss of CD133(+) CSCs. Taken together, our data indicate that dimerization of iCasp9 circumvents the apoptosis block in CSCs, which results in effective tumor regression in vivo.

Methylation of cancer-stem-cell-associated Wnt target genes predicts poor prognosis in colorectal cancer patients.

Gene signatures derived from cancer stem cells (CSCs) predict tumor recurrence for many forms of cancer. Here, we derived a gene signature for colorectal CSCs defined by high Wnt signaling activity, which in agreement with previous observations predicts poor prognosis. Surprisingly, however, we found that elevated expression of Wnt targets was actually associated with good prognosis, while patient tumors with low expression of Wnt target genes segregated with immature stem cell signatures. We discovered that several Wnt target genes, including ASCL2 and LGR5, become silenced by CpG island methylation during progression of tumorigenesis, and that their re-expression was associated with reduced tumor growth. Taken together, our data show that promoter methylation of Wnt target genes is a strong predictor for recurrence of colorectal cancer, and suggest that CSC gene signatures, rather than reflecting CSC numbers, may reflect differentiation status of the malignant tissue.

The cytotoxic protease granzyme M is expressed by lymphocytes of both the innate and adaptive immune system.

The cytotoxic serine protease granzyme M (GrM) is one of the five human granzymes, which are mainly expressed by cytotoxic T lymphocytes and/or NK cells. Upon perforin-dependent entry into a target cell, GrM cleaves specific substrates resulting in the onset of a unique cell death mechanism. However, the role of GrM in pathophysiological conditions is not clear yet. Knowledge of the expression and regulation of GrM by lymphocyte populations is instrumental for a better understanding of the contribution of this unique granzyme in health and disease. Two previous studies demonstrated GrM protein expression by lymphocytes of the innate immune system, i.e., NK cells, NKT cells, and gammadelta T cells, whereas its expression by CD8(+) T cells remained controversial. In the present study, we have investigated the expression and regulation of GrM in lymphocyte subsets in more detail. Flow cytometry analysis with a novel specific antibody against human GrM confirmed high expression of this protease by NK cells, NKT cells, and gammadelta T cells. CD8(+) T cells also expressed GrM and comparing the naive to early effector-memory, to late effector-memory, to effector subset, this expression gradually increased during differentiation. In contrast, CD4(+) T cells hardly expressed GrM. Quantitative PCR analysis for GrM mRNA levels in the diverse lymphocyte sub-populations confirmed the FACS results. GrM protein expression by lymphocyte populations was not significantly affected by a panel of GrB-inducing cytokines, indicating that GrM expression is differentially regulated as compared to GrB. In conclusion, the human cytotoxic protease GrM is, besides by innate immune cells, also expressed by CD8(+) effector T cells, in particular by the differentiated effector CD27(-) CD45RO(-) subset. Our current findings support not only a role for GrM in the innate but also in the adaptive immune response.